ICH-induced brain injury can be divided into two phases: primary injury caused by the mass effect of intraparenchymal hematoma and secondary injury caused by oxidative stress and neuroinflammation in the perihematomal area[17, 18].
Hematoma and its degradation products may activate post-ICH inflammatory responses in the perihematomal region[19]. Effective hematoma removal is crucial for regulating inflammation and functional recovery[20]. Microglia are crucial for tissue repair involving hematoma and damaged-cell phagocytosis post-ICH; moreover, they exert anti-inflammatory and pro-inflammatory effects. Microglia are innate immune cells in the brain and are considered as the first kind of non-neuronal cells to respond to various acute brain injuries, including ICH [21, 22]. Therefore, this study established a microglia/RBC co-culture model in vitro to explore the microglial involvement in ICH.
Hematoma removal and absorption are essential for ICH recovery and are clinically achieved through craniotomy or minimally invasive hematoma removal surgery and drug treatment. Moreover, the quality of clinical outcomes is positively correlated with the speed of hematoma absorption[23]. Therefore, promoting endogenous hematoma absorption has become a novel ICH treatment strategy[24]. Microglia, which are effector cells of immune and inflammatory responses in the central nervous system, can clear hematomas and protect nerve cells by phagocytizing RBCs and dissolved RBC components. In the LPS-induced inflammatory injury model, the HO-1/CO system was found to promote microglia migration, accelerate microglial phagocytosis, and protect central nervous system[25]. In the microglia/erythrocyte co-culture model in this study, the microglia + PKH26+RBCs + CORM-2 group was confirmed have more strong efficacy on increasing HO-1and decreasing IL-1β and NF-κB p65. All of which indicated that CORM-2 could increase microglial phagocytosis of RBCs and inhibit bleeding-induced inflammation.
In addition to accelerating hematoma absorption, it is important to regulate the inflammatory response around the hematoma. Neuronal injury and related neurological outcomes are dependent on a delicate balance between pro-inflammatory and anti-inflammatory mediators[26]. Endogenous CO is mainly oxidized by heme, widely involved in cardiovascular diseases, respiratory lesions, and other physiological /pathophysiological processes, has been confirmed with anti-inflammatory, anti-apoptotic, and anti-oxidative effects[13, 27].
CO was confirmed could inhibit the expression of inflammatory factors, such as IL-1β, and macrophage inflammatory protein 1β, and reduce inflammatory injury[28, 29]. The HO-1/CO system can inhibit TNF-α-mediated inflammatory response [27], and CORM-2 can not only up-regulate HO-1 but also induce the cytoprotective effect of HO-1[30]. This study revealed the mechanism underlying erythrophagocytosis modulation by the HO-1/CO system and neuroprotection by microglia. Activated microglia can release reactive oxygen species, which cause protein oxidation, membrane lipid peroxidation, enzyme inactivation, and DNA damage [31]. However, Mayne reported that decreased microglial TNF-α expression reduced neuronal apoptosis around the hematoma and improved the neurobehavioral score[32]. Our findings confirmed that CORM-2 effectively increased HO-1 expression, as well as inhibited NF-κB p65 and IL-1β expression in the microglia and RBC co-culture model, which suggested that CORM-2 could induce the microglia anti-inflammatory effect. However, it remains unclear whether CORM-2 inhibits the NF-κB signaling pathway by suppressing NF-κB p65 subunit phosphorylation or the nuclear translocation process of the NF-κB p65 subunit. HO-1 could inhibit the production of numerous downstream inflammatory factors of NF-κB by inhibiting and promoting NF-κB p65 and nuclear factor 2-related factor (Nrf2) entry, respectively, into the nucleus. Therefore, it plays a neuroprotective effect on early injury around the cerebral hemorrhage focus in rats[8–10]. In this study, CORM-2 significantly inhibited cellular and nuclear NF-κB p65 expression, which indicates that the HO-1/CO system may enhance the microglia anti-inflammatory effect by inhibiting post-ICH nuclear translocation of NF-κB p65 subunit.
Finally, we assessed whether CORM2 increased HbCO and affected oxygen metabolism. The affinity of carbon monoxide to hemoglobin is approximately 200 times greater than that of oxygen; therefore, CO poisoning could cause hypoxic damage. CORM-2 can slowly release CO in DMSO solution, which is convenient for controlling the CO release rate[33]. We found that CORM-2 adding to the microglia + RBC co-culture model did not increase the HbCO levels; instead, it mildly decreased HbCO. CORM-2 could enhance microglial phagocytosis to RBCs, which decreased hemoglobin level. In this study, although there was a gradual increase in CO expression, HbCO saturation remained stable (500–600 ng/ml) after CORM-2 treatment, indicated that CORM-2 did not produce serious toxic and side effects. And also, CORM-2 did not result in excessive carboxyhemoglobin levels. Study identified that low level of HbCO did not insignificantly affect overall mitochondrial function and biogenetics, but resulted in a significant increasing in the basal oxygen consumption rate. Assessment of mitochondrial function with inhibitors revealed no other alterations in the oxygen consumption rate [34]. Although CO gas has already passed safety evaluation in phase I trials in healthy humans and possesses a backbone carrier moiety, CORM-2 should be stringently characterized from a metabolic and toxicological perspective[35]. Further study is needed to elucidate the pharmacokinetics and biology of CO and CORMs.